The present invention relates to neurotrophic and analgesic peptides and their methods of use. More specifically, the invention relates to stable, active retro-inverso analogs of neurotrophic peptide fragments of saposin C.
Demyelination is a defect common to a number of central nervous system (CNS) disorders, the most prevalent being multiple sclerosis (MS). MS, a chronic disorder which may lead to total disability, is characterized by damage to the myelin sheath, leaving the axons mostly intact. MS is the most prevalent neurological disease of young adults. The incidence of MS in the United States is approximately 300,000. Currently, the treatment for MS using anti-inflammatory drugs is palliative rather than curative; reversal of demyelination is minimal since these drugs act to reduce inflammation rather than promote repair (Interferon Therapy of Multiple Sclerosis, Reder, A. ed., Marcel Dekker, New York, 1997). Other central nervous system disorders involving demyelination include acute disseminated encephalomyelitis, trauma to brain and/or spinal cord, acute hemorrhagic leukodystrophy, progressive multifocal leukoencephalitis, metachromatic leukodystrophy, adrenal leukodystrophy and maldevelopment of the white matter in premature infants (periventricular leucomalacia). The peripheral nervous system (PNS) can also undergo demyelination, such as that occurring in Guillain-Barrxc3xa9 syndrome (Pathologic Basis of Disease, Robbins et al. eds., W. B. Saunders, Philadelphia, 1979, pp. 1578-1582), traumatic injury and inflammatory or infectious neuropathies. Peripheral nerve injuries and peripheral neuropathies, such as those resulting from diabetes or chemotherapy, comprise the most prevalent peripheral nervous system disorders.
Neurotrophins and neurotrophic factors are proteins or peptides capable of affecting the survival, target innervation and/or function of neuronal cell populations (Barde, Neuron, 2:1525-1534, 1989). The efficacy of neurotrophins both in vivo and in vitro has been well documented. For example, nerve growth factor (NGF) acts as a trophic factor for forebrain cholinergic, peripheral and sensory neurons (Hefti et al., Neurobiol. Aging, 10:515-533, 1989). In vivo experiments indicate that NGF can reverse naturally-occurring as well as physical traumatic injuries to peripheral nerves (Rich et al., J. Neurocytol., 16:261-268, 1987). Brain-derived neurotrophic factor (BDNF) is a trophic factor for peripheral sensory neurons, dopaminergic neurons of the substantia nigra, central cholinergic neurons and retinal ganglia (Henderson et al., Restor. Neurol. Neurosci., 5:15-28, 1993). BDNF has been shown to prevent normally-occurring cell death both in vitro and in vivo (Hofer et al., Nature, 331:261-262, 1988). Ciliary neurotrophic factor (CNTF) promotes survival of chicken embryo ciliary ganglia in vitro and supports survival of cultured sympathetic, sensory and spinal motor neurons (Ip et al., J. Physiol. Paris, 85:123-130, 1991).
Prosaposin is the precursor of a group of four small heat-stable glycoproteins which are required for hydrolysis of glycosphingolipids by lysosomal hydrolases (Kishimoto et al., J. Lipid Res. 33:1255-1267, 1992). Prosaposin is proteolytically processed in lysosomes, generating saposins A, B, C and D (O""Brien et al., FASEB J., 5:301-308, 1991). O""Brien et al. (Proc. Natl. Acad. Sci. U.S.A., 91:9593-9596, 1994), U.S. Pat. No. 5,571,787 and published PCT Application No. WO95/03821, the entire contents of which are hereby incorporated by reference, disclose that prosaposin and saposin C stimulate neurite outgrowth and promote increased myelination. In addition, U.S. Pat. No. 5,571,787 and WO95/03821 disclose that a 22-mer peptide (CEFLVKEVTKLIDNNKTEKEIL; SEQ ID NO: 1) consisting of amino acids 8-29 of human saposin C stimulates neurite outgrowth in both neuroblastoma cells and mouse cerebellar explants. These references also disclose that an 18-mer peptide (YKEVTKLIDNNKTEKEIL; SEQ ID NO: 2) contained within the active 22-mer of saposin C (with V replaced by Y) also promotes neurite outgrowth and was able to cross the blood-brain barrier. O""Brien et al. (FASEB J., 9:681-685, 1995) show that the 22-mer stimulates choline acetyltransferase activity and prevented cell death in neuroblastoma cells in vitro. The active neuritogenic fragment was localized to a linear 12-mer located in the amino-terminal sequence of saposin C (LIDNNKTEKEIL; SEQ ID NO: 3). The 22-mer (SEQ ID NO: 1) is a loop at the adjacent asparagine residues flanked by helical regions in native prosaposin.
A major obstacle to the in vivo therapeutic use of peptides is their susceptibility to proteolytic degradation. Retro-inverso peptides are isomers of linear peptides in which the direction of the sequence is reversed (retro) and the chirality, D or L, of each amino acid is inverted (inverso). There are also partially modified retro-inverso isomers of linear peptides in which only some of the peptide bonds are reversed and the chirality of the amino acid residues in the reversed portion is inverted. The major advantage of such peptides is their enhanced activity in vivo due to improved resistance to proteolytic degradation (For review, see Chorev et al., Trends Biotech., 13:438-445, 1995). Although such retro-inverso analogs exhibit increased metabolic stability, their biological activity is often greatly compromised (Guichard et al., Proc. Natl. Acad Sci. U.S.A., 91:9765-9769, 1994). For example, Richman et al. (J. Peptide Protein Res., 25:648-662) determined that analogs of linear and cyclic leu-enkephalin modified at the Gly3-Phe4 amide bond had activities ranging from 6%-14% of native leu-enkephalin. Chorev et al., (ibid.) showed that retro-inversion of a peptide which inhibits binding of vitronectin to its receptor resulted in one peptide which was less potent than the parent isomer by a factor of 50,000, and another peptide which was 4,000 fold more potent than the parent cyclic peptide. Guichard et al. (TIBTECH 14, 1996), teach that retro-inverso (all-D-retro) antigenic mimicry may only occur with peptides in random coil, loop or cyclic conformations. In the case of xe2x80x9chelicalxe2x80x9d peptide, adequate functional mimicry would be expected only if the helicity was, in fact, absent under the solvent conditions used for asessing antigenic mimicry.
There is a need for neurotrophic and analgesic peptides exhibiting increased metabolic stability while retaining biological activity. The present invention addresses this need.
The present invention includes the discovery that retro-inverted peptides of neurotrophic saposin C fragments have a surprisingly high level of neurotrophic activity. Modified and substituted versions of these peptides are also surprisingly active, and are stable in vivo. Simple screening protocols are set forth hereinbelow to enable rapid determination of activity for any particular candidate peptide. Although retro-inverted peptides have, in the past, had poor activity, the present invention illustrates that the retro-inverso approach is ideally suited for neurotrophic fragments of saposin C.
One embodiment of the present invention is a peptide having between 10 and about 40 amino acids, wherein the peptide includes the amino acid sequence shown in SEQ ID NO: 12. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5, 7, 8 or 11. Preferably, the peptide is modified at the amino terminus, carboxy terminus, or both amino and carboxy terminus with one of the following independently selected moieties: CH3CO, CH3(CH2)nCO, C6H5CH2CO and H2N(CH2)nCO, wherein n=1-10. Advantageously, the peptide is glycosylated at Asn7 or at the alpha amino group. In one aspect of this preferred embodiment, one or more amide bonds of the peptide is reduced. In another aspect of this preferred embodiment, one or more nitrogens in the peptide is methylated. Preferably, one or more carboxylic acid groups in the peptide is esterified.
The present invention also provides a method for stimulating neuritogenesis or preventing neural cell death, comprising the step of contacting neural cells with a composition comprising an effective neuritogenic or neural cell death-preventing amount of a peptide having between 11 and about 40 amino acids, wherein the peptide includes the amino acid sequence shown in SEQ ID NO: 12. Preferably, the neuronal cells are neuroblastoma cells. Advantageously, the neuroblastoma cells are NS20Y cells.
Another embodiment of the present invention is a method for stimulating myelination or preventing demyelination, comprising the step of contacting neural cells with a composition comprising an effective myelin-stimulating or demyelination-inhibiting amount of a peptide having between 11 and about 40 amino acids, wherein said peptide includes the amino acid sequence shown in SEQ ID NO: 12. Preferably, the peptide has the sequence selected shown in SEQ ID NOS: 5, 7, 8 or 11.
The present invention also provides a method for treating pain in a mammal in need thereof, comprising administering to the mammal an effective pain-treating amount of a composition comprising a peptide having between 11 and about 40 amino acids, wherein the peptide includes the amino acid sequence shown in SEQ ID NO: 12. Preferably, the peptide has the sequence shown in SEQ ID NOS: 5, 7, 8 or 11.
The present invention also provides a pharmaceutical composition comprising a peptide having between 11 and about 40 amino acids, wherein the peptide includes the sequence shown in SEQ ID NO: 12, in a pharmaceutically acceptable carrier. Preferably, the composition is in a controlled release formulation. Alternatively, the composition is in liposomal form. The composition may also be in lyophilized form. Preferably, the composition is in unit dosage form.
The present invention also provides a method for stimulating myelination or inhibiting demyelination in a mammal in need thereof, comprising the step of administering to the mammal a composition comprising an effective myelin-stimulating or demyelination-inhibiting amount of a peptide having between 10 and about 40 amino acids, wherein the peptide includes the amino acid sequence shown in SEQ ID NO: 12. preferably, the peptide has the sequence shown in SEQ ID NOS: 5, 7, 8 or 11. Advantageously, the administering step comprises intravenous, pulmonary, intrathecal, intramuscular, intradermal, subcutaneous, intracranial, epidural, topical and oral.